Methods and means for modulating PGE synthase activity

ABSTRACT

Isolated PGE synthase, provided from encoding nucleic acid. Methods of production and use. Assays for modulators, especially inhibitors, of PGE synthase activity.

This application is a divisional of U.S. Pat. application Ser. No.09/433,466, filed Nov. 4, 1999, now U.S. Pat. No. 6,395,502, whichclaims priority under 35 U.S.C. §119(e) to U.S. Provisional Application60/107,687 filed Nov. 9, 1998.

The present invention relates to modulating PGE synthase activity. Inparticular, the present invention is based on identification of PGEsynthase and DNA encoding it, providing for assays for substances ableto modulate, especially inhibit, PGE synthase activity. PGE is a potentcompound known to produce inflammation (symptoms including fever andpain), and inhibition of its production may be used in treatment ofinflammation, arthritis, cancer, Alzheimer's disease, in modulatingapoptosis, and treating pain.

Prostaglandin endoperoxide H₂ (PGH₂) is formed from arachidonic acid bythe action of cyclooxygenase (cox) -1 or -2. Cox-1 is constitutivelyexpressed in many cells and tissues such as platelets, endothelium,stomach and kidney whereas the cox-2 protein can be induced byproinflammatory cytokines like interleukin-1β at sites of inflammation.For recent reviews on cox see Smith, W. (1997) Advances in ExperimentalMedicine & Biology 400B, 989-1011; Herschman, H. R. (1996) Biochimica etBiophysica Acta 1299, 125-40; Dubois, R., et al.(1998) Faseb J. 12,1063-1073. Downstream of the cyclooxygenases, their product PGH₂ can befurther metabolized into the various physiologically importanteicosanoids e.g. PGF_(2α), PGE₂, PGD₂, PGI₂ (prostacyclin) andthromboxane (TX) A₂ (Smith, W. L. (1992) Am. J. Physiol. 263,F181-F191).

The mechanism for the biosynthesis of PGE₁ and PGF_(1α) (formed usingdihomo-γ-linolenic acid instead of arachidonic acid) (Hamberg, M. &Samuelsson, B. (1967) J. Biol. Chem. 242, 5336-5343) by sheep vesicularglands was postulated to proceed via a cyclic endoperoxide (Samuelsson,B. (1965) J. Am. Chem. Soc. 87, 3011-3013) later designated PGH2(Hamberg, M. & Samuelsson, B. (1973) Proc. Natl. Acad. Sci. USA 70,899-903; Hamberg, M., et al. (1974) Proc. Natl. Acad. Sci. USA 71,345-349; Nugteren, D. H. & Hazelhof, E. (1973) Biochim. Biophys. Acta326, 448-461). In short, the reactions catalyzed by the cyclooxygenasesinvolve a stereospecific abstraction of the 13-pro-S hydrogen atom fromarachidonic acid. This leads to the formation of a carbon radical thatis trapped by molecular oxygen at position C-11, formation of the9,11-endoperoxide and the bond between the C-8 and C-12 positions withtrans aliphatic side chains, radical rearrangement to C-15 and reactionwith a second molecule of oxygen. In the next step the resulting peroxygroup at C-15 is reduced to a hydroperoxy group and PGG₂ is formed. Thishydroperoxy group can subsequently be reduced by the peroxidase activityof cyclooxygenase (in the presence of a reducing agent e.g. glutathione)thus forming PGH₂ The enzyme/s responsible for the isomerization of PGH₂into PGE₂ are not well known. Attempts have been made to purify themicrosomal PGE synthase from ovine and bovine seminal vesicles, an organknown to contain high PGE synthase activity (Ogino, N., et al. (1977)Journal of Biological Chemistry 252, 890-5; Moonen, P., et al. (1982)Methods in Enzymology 86, 84-91). These studies have shown that themicrosomal PGE synthase can be solubilized and partly purified. Theenzyme activity was also dependent on glutathione but rapidlyinactivated during the course of purification. Two monoclonal antibodiesdesignated IGGl(hei-7) and IGGl(hei-26) raised against partly purifiedPGE synthase from sheep seminal vesicles, could immunoprecipitate twoproteins from sheep seminal vesicles with molecular masses of 17.5 and180 kDa, respectively (Tanaka, Y., et al. (1987) J. Biol. Chem. 262,1374-1381). Both these precipitated proteins were found to possessglutathione dependent PGE synthase activity but no glutathioneS-transferase activity. Interestingly, the IGG1(hei-7) antibody alsocaused co-precipitation of cyclooxygenase, demonstrating that the 17.5kDa protein and the cox proteins were on the same side of the microsomalmembranes. The 17.5 kDa protein showed a Km for PGH₂ of 40 μM, similarto what has been described by others investigating the microsomal PGEsynthase (Moonen, P., et al. (1982) Methods in Enzymology 86, 84-91). Incontrast, the larger protein demonstrated a Km for PGH₂ of 150 μM.Additional proteins, belonging to the cytosolic glutathioneS-transferase superfamily, have also been described to possess PGE, PGDand PGF synthase activities (Urade, Y., et al. (1995) J. Lipid Med. 12,257-273). Recently, a microsomal 16.5 kDa protein was purified fromsheep seminal vesicles possessing glutathione dependent PGF_(2α)synthase activity (Burgess, J. R. & Reddy, C. C. (1997) Biochem. & Mol.Biol. Int. 41, 217-226). The enzyme (prostaglandin endoperoxidereductase) could also catalyze the reduction of cumene hydroperoxidewhereas, 1-chloro-2,4-dinitrobenzene (typical substrate for variousglutathione S-transferases) was not a substrate. Microsomal PGE synthaseactivity was also measured in various rat organs (Watanabe, K., etal.(1997) Biochemical & Biophysical Research Communications 235, 148-52)and high glutathione dependent activity was found in the deferens duct,genital accessory organs and kidney. Glutathione independent microsomalPGE synthase activity was observed in heart, spleen and uterus.

The enzyme responsible for PGE biosynthesis therefore provides a noveltarget for drug development in order to treat various inflammatorydisorders. However, as is apparent from the preceding discussion, no-onehas previously succeeded in providing pure PGE synthase nor the means toprovide it.

Oxford Biomedical sells a partially purified preparation of ovine PGEsynthase (Catalog Number PE 02). Analysis of that preparation indicatesit is rather crude, including a complex mixture of numerous components.

Particular difficulties in purifying PGE synthase include the fact thatthe protein is a membrane protein, in general very hard to purify tohomogeneity and the fact that its enzyme activity is very unstable aftersolubilization. Also, the work described herein demonsrates that theprotein possesses very high enzyme activity, providing indication thatthe amounts of protein are very low within cells, adding to thedifficulty of purification.

Urade et al.(1995) J. Lipid Med. 12, 257-273, notes “little is knownabout the properties of PGE synthase”. Even more recently, William Smithin “Molecular Biology of Prostanoid Biosynthetic Enzymes and Receptors”,Advances in Experimental Medicine & Biology, 400B: 989-1011, publishedin 1997, noted “The PGE synthase story has been a perplexing one”,pointing out that PGE formation has not been attributed to a uniqueprotein.

The work of the present inventors described below demonstrates thathuman PGE synthase is a member of a protein superfamily consisting ofmembrane associated 14-18 kDa proteins involved in eicosanoid andglutathione metabolism. PGE synthase demonstrates 38% identity on theamino acid sequence level with microsomal glutathione S-transferase 1.The human cDNA sequence as well as the predicted amino acid sequencewere deposited in 1997 in public databases under the name of MGST1-L1(GenBank accession number AF027740) as well as a p53 induced PIG12(GenBank accession number AF010316). No function has previously beenascribed to these cDNA sequences.

Polyak et al. (1997) Nature 389: 300-305 identified what they called“PIG12” by cloning sequences of which expression was upregulated by P53.They state “PIG12 is a novel member of the microsomal glutathioneS-transferase family of genes”, but identify not actual function. Thereis certainly no suggestion that their PIG12 was actually human PGEsynthase.

In summary, no-one has previously provided PGE synthase in any form orquantity that would allow for amino acid sequencing to provide apotential starting point for attempted cloning of a coding sequence.Furthermore there was no suggestion that the sequence on the databaseswhich the present inventors have now demonstrated to encode PGE synthasedid actually encode PGE synthase.

In the light of the inventors' work, the present invention provides invarious aspects for use of purified PGE synthase in various contexts, inparticular in assays and screening methods for substances able tomodulate, especially inhibit, PGE synthase activity. The purified PGEsynthase may be made by recombinant expression from encoding nucleicacid. It may be expressed in eukaryotic or prokaryotic expressionsystems and may lack native glycosylation. Substances identified asmodulators of PGE synthase may be employed in control or treatment ofinflammation, arthritis, cancer or other cellular growth abnormality,Alzheimer's disease, in modulating apoptosis, and treating pain.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the results of reverse-phase HPLC chromatogram of theproducts formed after incubations with PGH₂ (plotting counts per minute(CPM) against time in minutes).

FIG. 1A shows results obtained with PGE synthase membrane fraction mixedwith stop solution.

FIG. 1B shows results obtained with buffer.

FIG. 1C shows results obtained with PGE synthase membrane fraction. Band C were incubated for 2 min prior to addition of stop solution.Products were detected using radioactivity detection. The first 20 minrepresents isocratic elution using water, acetonitrile andtrifluoroacetic acid (70:30:0.007, by vol) as mobile phase with a flowrate of 1 ml/min. Then a linear gradient was applied from 100% mobilephase to 100% methanol over a 10 min period, which was sustained for therest of the run.

FIG. 2 illustrates dependency of PGE₂ formation on membrane proteinconcentration, amount of PGE₂ in pmol being plotted against mg/ml of theprotein.

FIG. 3 shows a time course for PGE₂ formation, amount of PGE₂ in pmolbeing plotted against time in minutes. Filled circles are for PGEsynthase incubated with glutathione; open circles are for PGE synthasewithout glutathione; filled triangles are for buffer with glutathione.

The following abbreviations are used herein:

PGG₁, Prostaglandin G₁: 15(S)-hydroperoxy-9α,11α-peroxidoprosta-13-enoicacid;

PGG₂, Prostaglandin G₂:15(S)-hydroperoxy-9α,11α-peroxidoprosta-5-cis-13-trans-dienoic acid;

PGG₃, Prostaglandin G₃:15(S)-hydroperoxy-9α,11α-peroxidoprosta-5,13,17-trienoic acid;

PGH₁, Prostaglandin H₁: 15(S)-hydroxy-9α,11α-peroxidoprosta-13-enoicacid;

PGH₂, Prostaglandin H₂:15(S)-hydroxy-9α,11α-peroxidoprosta-5-cis-13-trans-dienoic acid;

PGH₃, Prostaglandin H₃:15(S)-hydroxy-9α,11α-peroxidoprosta-5,13,17-trienoic acid;

PGE₂, Prostaglandin E₂: 11α,15(S)-dihydroxy-9-ketoprosta-5-cis-13-trans-dienoic acid;

PGF_(2α), Prostaglandin F_(2α):9α,11α,15(S)-trihydroxyprosta-5-cis-13-trans-dienoic acid;

PGD₂, Prostaglandin D₂: 9α,15(S)-dihydroxy-11-ketoprosta-5-cis-13-trans-dienoic acid;

PGI₂, Prostacyclin:6,9α-epoxy-11α,15(S)-dihydroxyprosta-5-cis-13-trans-dienoic acid;

TXA₂, Thromboxane A₂:9α,11α,epoxy-15(S)-hydroxythromba-5-cis-13-trans-dienoic acid;

12-HHT: 12(S)-Hydroxy-8,10-trans-5-cis-heptadecatrienoic acid; PGHsynthase: Prostaglandin H synthase;

RP-HPLC: Reverse-phase high performance liquid chromatography;

LT: Leukotriene;

LTA₄, Leukotriene A₄:5(S)-trans-5,6-oxido-7,9-trans-11,14-cis-eicosatetraenoic acid;

LTC₄, Leukotriene C₄:5(S)-hydroxy-6(R)-S-glutathionyl-7,9-trans-11,14-cis-eicosatetraenoicacid;

FLAP: 5-lipoxygenase activating protein;

MGST: microsomal glutathione S-tranferase;

NSAID: Nonsteroidal anti-inflammatory drugs.

The present invention provides pure PGE synthase. A preferredpolypeptide of the invention includes the amino acid sequence of SEQ IDNO. 2.

Isolated polypeptides of the invention will be those as defined hereinin isolated form, free or substantially free of material with which itis naturally associated such as other polypeptides with which it isfound in the cell. The polypeptides may of course be formulated withdiluents or adjuvants and still for practical purposes be isolated—forexample the polypeptides will normally be mixed with gelatin or othercarriers if used to coat microtitre plates for use in immunoassays. Thepolypeptides may be glycosylated, either naturally or by systems ofheterologous eukaryotic cells, or they may be (for example if producedby expression in a prokaryotic cell) unglycosylated. The term “lackingnative glycosylation” may be used with reference to a polypeptide whicheither has no glycosylation (e.g. following production in a prokaryoticcell) or has a pattern of glycosylation that is not the native pattern,e.g. as conferred by expression in a particular host cell type (whichmay be CHO cells).

Polypeptides of the invention may be modified for example by theaddition of a signal sequence to promote their secretion from a cell orof histidine residues to assist their purification. Fusion proteins maybe generated that incorporate (e.g.) six histidine residues at eitherthe N-terminus or C-terminus of the recombinant protein. Such ahistidine tag may be used for purification of the protein by usingcommercially available columns which contain a metal ion, either nickelor cobalt (Clontech, Palo Alto, Calif., USA). These tags also serve fordetecting the protein using commercially available monoclonal antibodiesdirected against the six histidine residues (Clontech, Palo Alto,Calif., USA).

Polypeptides which are amino acid sequence variants, alleles,derivatives or mutants are also provided by the present invention, suchforms having at least 70% sequence identity, for example at least 80%,90%, 95%, 98% or 99% sequence identity to SEQ ID NO. 2. A polypeptidewhich is a variant, allele, derivative or mutant may have an amino acidsequence which differs from that given in SEQ ID NO. 2 by one or more ofaddition, substitution, deletion and insertion of one or more (such asfrom 1 to 20, for example 2, 3, 4, or 5 to 10) amino acids.

The amino acid sequence of SEQ ID NO. 2 is encoded by the humannucleotide sequence of SEQ ID NO. 1. Polypeptides of the inventioninclude those encoded by alleles of the human sequence, and homologuesof other mammals, particularly primates, as well as fragments of suchpolypeptides as discussed further below. The primary sequence of the PGEsynthase protein will be substantially similar to that of SEQ ID NO. 2and may be determined by routine techniques available to those of skillin the art. In essence, such techniques include using polynucleotidesderived from SEQ ID NO. 1 as probes to recover and to determine thesequence of the PGE synthase gene in other species. A wide variety oftechniques are available for this, for example PCR amplification andcloning of the gene using a suitable source of mRNA, or by methodsincluding obtaining a cDNA library from the mammal, e.g a cDNA libraryfrom one of the above-mentioned sources, probing said library with apolynucleotide of the invention under stringent conditions, andrecovering a cDNA encoding all or part of the PGE synthase protein ofthat mammal. Where a partial cDNA is obtained, the full length codingsequence may be determined by primer extension techniques.

An “active portion” of the polypeptides means a peptide which is lessthan said full length polypeptide, but which retains its essentialbiological activity. In particular, the active portion retains theability to catalyse PGE synthesis from PGH in the presence ofglutathione.

Suitable active portions thus include the central segment of SEQ ID NO.2, e.g. between about residues 30-130. The relevant catalytic region ofthe PGE synthase protein is expected to be in the central segment of SEQID NO. 2 based on analogy with MGST1 and LTC₄ synthase: amino acids 1-41can be removed from MGST1 by proteolysis without loss of function(Andersson et al., (1994) Biochim. Biophys. Acta 1204, 298-304);C-terminal segments can be exchanged between LTC₄ synthase and FLAPwithout alteration of protein function (Lam et al., (1997) J. Biol.Chem. 272, 13923-13928).

One active portion of the invention includes or consists of amino acids30-152 of SEQ ID NO. 2. Another active portion includes or consists ofamino acids 1-130 of SEQ ID NO. 2. A still further active portionincludes or consists of amino acids 30-130 of SEQ ID NO. 2.

The present invention includes a polypeptide including an active portionof a PGE synthase provided herein, which polypeptide may includeheterologous amino acids, such as an identifiable sequence or domain ofanother protein, or a histidine tag or other tag sequence, and theinvention includes a polypeptide consisting essentially of an activeportion of a PGE synthase.

A polypeptide according to the present invention may be isolated and/orpurified (e.g. using an antibody) for instance after production byexpression from encoding nucleic acid. Polypeptides according to thepresent invention may also be generated wholly or partly by chemicalsynthesis, for example in a step-wise manner. The isolated and/orpurified polypeptide may be used in formulation of a composition, whichmay include at least one additional component, such as a diluent.

A polypeptide according to the present invention may be used inscreening for molecules which affect or modulate its activity orfunction. Such molecules may be useful in a therapeutic (which mayinclude prophylactic) context. This is discussed in detail below.

A polypeptide of the invention may be labelled with a revealing label.The revealing label may be any suitable label which allows thepolypeptide to be detected. Suitable labels include radioisotopes, e.g.¹²⁵I, enzymes, antibodies, polynucleotides and linkers such as biotin.

As noted, a preferred way of producing a polypeptide of the invention isto employ encoding nucleic acid in a suitable expression system toproduce the polypeptide recombinantly. In a further aspect the presentinvention provides the use of nucleic acid encoding PGE synthasepolypeptide in production of PGE synthase.

Nucleic acids of the present invention include nucleic acids whichinclude a sequence encoding a polypeptide which includes the amino acidsequence of SEQ ID NO. 2 and a polypeptide having at least 70% sequenceidentity to SEQ ID NO. 2. Preferably the degree of sequence identity ineither case is at least 80%, such as at least 90%, 95%, 98% or 99%.

Nucleic acids useful in the invention further include nucleic acidswhich include a sequence having at least 70% homology, more preferablyat least 80%, such as at least 90%, 95%, 98% or 99% sequence homology tothe nucleic acid sequences of SEQ ID NO. 1 or its complement.

Nucleic acid of the invention may encode the amino acid sequence of SEQID NO. 2, in which case it may include SEQ ID NO. 1 or a differentnucleotide sequence, as permitted by degeneracy of the genetic code, ora polypeptide with PGE synthase activity which has an amino acidsequence which differs from SEQ ID NO. 2.

Where an aspect of the present invention is expressed in terms ofnucleic acid with at least a specified % homology with SEQ ID NO. 1 orits complement, the actual sequence of SEQ ID NO. 1 or its complementmay be excluded. In various embodiments the present invention providesnon-naturally occurring nucleic acid encoding a polypeptide PGE synthaseactivity, such as a polypeptide including the amino acid sequence of SEQID NO. 2 or an allelic variant thereof, or a non-naturally occurringpolypeptide mutant, variant or derivative thereof.

Nucleic acid sequences encoding all or part of a PGE synthase gene canbe readily prepared by the skilled person using the information andreferences contained herein and techniques known in the art (forexample, see Sambrook, Fritsch and Maniatis, “Molecular Cloning, ALaboratory Manual, Cold Spring Harbor Laboratory Press, 1989, andAusubel et al, Short Protocols in Molecular Biology, John Wiley andSons, 1992). These techniques include (i) the use of the polymerasechain reaction (PCR) to amplify samples of such nucleic acid, e.g. fromgenomic sources, (ii) chemical synthesis, or (iii) preparing cDNAsequences. Modifications to the wild type sequences described herein canbe made, e.g. using site directed mutagenesis, to lead to the expressionof modified polypeptides or to take account of codon preference in thehost cells used to express the nucleic acid.

In general, short sequences for use as primers will be produced bysynthetic means, involving a step wise manufacture of the desirednucleic acid sequence one nucleotide at a time. Techniques foraccomplishing this using automated techniques are readily available inthe art.

Longer polynucleotides will generally be produced using recombinantmeans, for example using a PCR (polymerase chain reaction) cloningtechniques. This will involve making a pair of primers (e.g. of about15-50 nucleotides) based on the sequence information provided herein toa region of the mRNA or genomic sequence encoding the mRNA which it isdesired to clone, bringing the primers into contact with mRNA or cDNAobtained from mammalian cells (which may for example be any of the humancell line A549, epithelial cells, osteosarcoma derived cell lines,osteoblasts, human leukocytes, fibroblasts, endothelial cells, cells ofthe reproductive system, mesangial cells and other kidney cells),performing a polymerase chain reaction under conditions which bringabout amplification of the desired region, isolating the amplifiedfragment (e.g. by purifying the reaction mixture on an agarose gel) andrecovering the amplified DNA. The primers may be designed to containsuitable restriction enzyme recognition sites so that the amplified DNAcan be cloned into a suitable cloning vector.

Such techniques may be used to obtain all or part of the sequencesdescribed herein. Genomic clones containing the PGE synthase gene andits introns and promoter regions may also be obtained in an analogousmanner, starting with genomic DNA from a mammalian, e.g. human cell,e.g. a primary cell such as a liver cell, a tissue culture cell or alibrary such as a phage, cosmid, YAC (yeast artificial chromosome), BAC(bacterial artificial chromosome) or PAC (P1/P2 phage artificialchromosome) library.

Polynucleotides which are not 100% homologous to the sequences of thepresent invention but fall within the scope of the invention can beobtained in a number of ways.

Other human variants (for example allelic forms) of the PGE synthasegene described herein may be obtained for example by probing cDNA orgenomic DNA libraries made from human tissue.

In addition, other animal, and particularly mammalian (e.g. mouse, rator rabbit, sheep, goat, cow, horse, pig, dog, cat, or primate)homologues of the gene may be obtained. Such sequences may be obtainedby making or obtaining cDNA libraries made from dividing cells ortissues or genomic DNA libraries from other animal species, and probingsuch libraries with probes including all or part of a nucleic acid ofthe invention under conditions of medium to high stringency (for examplefor hybridization on a solid support (filter) overnight incubation at42° C. in a solution containing 50% formamide, 5×SSC (750 mM NaCl, 75 mMsodium citrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution,10% dextran sulphate and 20 μg/ml salmon sperm DNA, followed by washingin 0.03 M sodium chloride and 0.03 M sodium citrate (i.e. 0.2×SSC) atfrom about 50° C. to about 60° C.).

Thus the present invention may employ an isolated nucleic acid whichhybridizes to the nucleotide sequence set forth in SEQ ID NO. 1 underthe abovementioned hybridization and washing conditions. Such a nucleicacid is suitable for use as a probe for detecting the a PGE synthasegene, for example in Southern blots or in metaphase spreads.

Alternatively, such polynucleotides may be obtained by site directedmutagenesis of the sequences of SEQ ID NO. 1 or allelic variantsthereof. This may be useful where for example silent codon changes arerequired to sequences to optimise codon preferences for a particularhost cell in which the polynucleotide sequences are being expressed.Other sequence changes may be desired in order to introduce restrictionenzyme recognition sites, or to alter the property or function of thepolypeptides encoded by the polynucleotides. Further changes may bedesirable to represent particular coding changes which are required toprovide, for example, conservative substitutions.

In the context of cloning, it may be necessary for one or more genefragments to be ligated to generate a full-length coding sequence. Also,where a full-length encoding nucleic acid molecule has not beenobtained, a smaller molecule representing part of the full molecule, maybe used to obtain full-length clones. Inserts may be prepared frompartial cDNA clones and used to screen cDNA libraries. The full-lengthclones isolated may be subcloned into expression vectors and activityassayed by transfection into suitable host cells, e.g. with a reporterplasmid.

Preferably, a polynucleotide of the invention in a vector is operablylinked to a control sequence which is capable of providing for theexpression of the coding sequence by the host cell, i.e. the vector isan expression vector. The term “operably linked” refers to ajuxtaposition wherein the components described are in a relationshippermitting them to function in their intended manner. A control sequence“operably linked” to a coding sequence is ligated in such a way thatexpression of the coding sequence is achieved under condition compatiblewith the control sequences.

Suitable vectors can be chosen or constructed, containing appropriateregulatory sequences, including promoter sequences, terminatorfragments, polyadenylation sequences, enhancer sequences, marker genesand other sequences as appropriate. Vectors may be plasmids, viral e.g.phage, phagemid or baculoviral, cosmids, YACs, BACs, or PACs asappropriate.

The vectors may be provided with an origin of replication, optionally apromoter for the expression of the said polynucleotide and optionally aregulator of the promoter. The vectors may contain one or moreselectable marker genes, for example an ampicillin resistance gene inthe case of a bacterial plasmid or a neomycin resistance gene for amammalian vector. Vectors may be used in vitro, for example for theproduction of RNA or used to transfect or transform a host cell. Thevector may also be adapted to be used in vivo, for example in methods ofgene therapy. Systems for cloning and expression of a polypeptide in avariety of different host cells are well known. Suitable host cellsinclude bacteria, eukaryotic cells such as mammalian and yeast, andbaculovirus systems. Mammalian cell lines available in the art forexpression of a heterologous polypeptide include Chinese hamster ovarycells, HeLa cells, baby hamster kidney cells, COS cells and many others.

For further details see, for example, Molecular Cloning: a LaboratoryManual: 2nd edition, Sambrook et al., 1989, Cold Spring HarborLaboratory Press. Many known techniques and protocols for manipulationof nucleic acid, for example in preparation of nucleic acid constructs,mutagenesis, sequencing, introduction of DNA into cells and geneexpression, and analysis of proteins, are described in detail in CurrentProtocols in Molecular Biology, Ausubel et al. eds., John Wiley & Sons,1992.

Vectors may be transformed into a suitable host cell as described aboveto provide for expression of a polypeptide of the invention. Thus, in afurther aspect the invention provides a process for preparingpolypeptides according to the invention which includes cultivating ahost cell transformed or transfected with an expression vector asdescribed above under conditions to provide for expression by the vectorof a coding sequence encoding the polypeptides, and recovering theexpressed polypeptides. Polypeptides may also be expressed in in vitrosystems, such as reticulocyte lysate.

Following production of a polypeptide of the invention it may be testedfor PGE synthase activity, e.g. by determination of PGE production onincubation of the polypeptide with PGH₂ and reduced glutathione. PGE canbe detected using Reverse-phase high pressure liquid chromatography (R-PHPLC) which allows for quantitation of the amount present.

Isolated/pure PGE synthase may be used in a variety of contexts.

Because of the importance of PGE in inflammation and other contexts ofmedical significance, important aspects of the invention are concernedwith identifying substances which are able to affect PGE production, inparticular by modulating PGE synthase activity. Of most interest is theinhibition of FGE synthase activity to reduce levels of PGE production.

PGE is well known to cause pain both in vivo and in vitro (Bley et al.(1998) Trends in Pharmacological Sciences 19, 141-147). Theprostaglandin E receptor (EP₃) has also been demonstrated to be crucialfor functional fever response (Ushikubi et al (1998) Nature 395,281-284). The role of prostaglandins in inflammation and inflammatorydiseases such as arthritis has also been well documented through the useof various cyclooxygenase inhibitors (nonsteroidal antioinflammatorydrugs, NSAIDs, including aspirin (Vane & Botting (1998) American J. ofMed. 104(3A), 2S-8S). In this respect, PGE has been recognized as themost potent proinflammatory prostaglandin (Moncada et al. (1973) Nature246, 217-9), which is which specific removal of this compound byinhibition of PGE synthase may be used to provide control ofinflammatory reaction with fewer side effects in comparison with thepresently used NSAIDs.

Several reports have demonstrated significant anti-tumour effects byNSAIDs on colorectal cancer (Giovannucci et al. (1994) Annals ofInternal Medicine 121, 241-6; Giardiello et al, (1995) European Journalof Cancer 31A, 1071; Williams et al (1997) Journal of Clinicalinvestigation 100, 1325-9). PGE promotes cancer cell proliferation (Qiaoet al (1995) Biochimica et Biophysica Acta 1258, 215-23) as well asinhibiting programmed cell death (Ottonello et al (1998) ExperimentalHematology 26, 895-902; Goetzl et al (1995) Journal of Immunology 154,1041-7), overall resulting in support of cancer cell growth (Sheng et al(1998) Cancer Research 58, 362-6). Inhibition of PGE formation thusleads to slower proliferation in combination with increased apoptosis ofthe cancer cell population. This inhibiting effect of NSAIDs has alsobeen observed in other cancer conditions such as non-small cell lungcancer (Hida et al. (1998) Anticancer Research 18, 775-82).

Prostaglandins have also been implicated in Alzheimer's disease. Severalclinical trials have demonstrated that users of NSAIDs experience aslittle as one half of the risk of acquiring Alzheimer's disease (Duboiset al (1998) Faseb J. 12, 1063-1073). Consistent with this, otherobservations suggest that inflammatory processes may contribute to thisdisease (Aisen (1997) Gerontology 43, 143-9).

In various further aspects the present invention relates to screeningand assay methods and means, and substances identified thereby,especially inhibitors of PGE synthase.

Thus, further aspects of the present invention provide the use of apolypeptide or peptide (particularly a fragment of a polypeptide of theinvention as disclosed, and/or encoding nucleic acid therefor), inscreening or searching for and/or obtaining/identifying a substance,e.g. peptide or chemical compound, which interacts and/or binds with thepolypeptide or peptide and/or interferes with its function or activityor that of another substance, e.g. polypeptide or peptide, whichinteracts and/or binds with the polypeptide or peptide of the invention.For instance, a method according to one aspect of the invention includesproviding a polypeptide or peptide of the invention and bringing it intocontact with a substance, which contact may result in binding betweenthe polypeptide or peptide and the substance. Binding may be determinedby any of a number of techniques available in the art, both qualitativeand quantitative.

In various aspects the present invention is concerned with provision ofassays for substances which interact with or bind a polypeptide of theinvention and/or modulate one or more of its activities.

One aspect of the present invention provides an assay which includes:

(a) bringing into contact a polypeptide or peptide according to theinvention and a putative binding molecule or other test substance; and

(b) determining interaction or binding between the polypeptide orpeptide and the test substance.

A substance which interacts with the polypeptide or peptide of theinvention may be isolated and/or purified, manufactured and/or used tomodulate its activity as discussed.

It is not necessary to use the entire proteins for assays of theinvention which test for binding between two molecules as above or testfor PGE synthase activity (see below). Fragments may be generated andused in any suitable way known to those of skill in the art. Suitableways of generating fragments include, but are not limited to,recombinant expression of a fragment from encoding DNA. Such fragmentsmay be generated by taking encoding DNA, identifying suitablerestriction enzyme recognition sites either side of the portion to beexpressed, and cutting out said portion from the DNA. The portion maythen be operably linked to a suitable promoter in a standardcommercially available expression system. Another recombinant approachis to amplify the relevant portion of the DNA with suitable PCR primers.Small fragments (e.g. up to about 20 or 30 amino acids) may also begenerated using peptide synthesis methods which are well known in theart.

The precise format of the assay of the invention may be varied by thoseof skill in the art using routine skill and knowledge. For example, theinteraction between the polypeptides may be studied in vitro bylabelling one with a detectable label and bringing it into contact withthe other which has been immobilised on a solid support. Suitabledetectable labels include ³⁵S-methionine which may be incorporated intorecombinantly produced peptides and polypeptides. Recombinantly producedpeptides and polypeptides may also be expressed as a fusion proteincontaining an epitope which can be labelled with an antibody.

The protein which is immobilized on a solid support may be immobilizedusing an antibody against that protein bound to a solid support or viaother technologies which are known per se. A preferred in vitrointeraction may utilise a fusion protein includingglutathione-S-transferase (GST). This may be immobilized on glutathioneagarose beads. In an in vitro assay format of the type described above atest compound can be assayed by determining its ability to diminish theamount of labelled peptide or polypeptide which binds to the immobilizedGST-fusion polypeptide. This may be determined by fractionating theglutathione-agarose beads by SDS-polyacrylamide gel electrophoresis.Alternatively, the beads may be rinsed to remove unbound protein and theamount of protein which has bound can be determined by counting theamount of label present in, for example, a suitable scintillationcounter.

Determination of the ability of a test compound to interact and/or bindwith a PGE synthase polypeptide or fragment may be used to identify thattest compound as a candidate for a modulator of PGE synthase activity.Generally, then identification of ability of a test compound to bind apolypeptide or fragment of the invention is followed by one or morefurther assay steps involving determination of whether or not the testcompound is able to modulate PGE synthase activity. Naturally, assaysinvolving determination of ability of a test substance to modulate PGEsynthase activity may be performed where there is no knowledge aboutwhether the test substance can bind or interact with the PGE synthase,but a prior binding/interaction assay may be used as a “coarse” screento test a large number of substances, reducing the number of candidatesto a more manageable level for a functional assay involvingdetermination of ability to modulate PGE synthase activity. A furtherpossibility for a coarse screen is testing ability of a substance toaffect PGE production by a suitable cell line expressing PGE synthase(either naturally or recombinantly). An assay according to the presentinvention may also take the form of an in vivo assay. The in vivo assaymay be performed in a cell line such as a yeast strain in which therelevant polypeptides or peptides are expressed from one or more vectorsintroduced into the cell. A still further possibility for a coarsescreen is testing ability of a substance to affect PGE production by animpure protein preparation including PGE synthase (whether human orother mammalian). Ultimately, however, a preferred assay of theinvention includes determining the ability of a test compound tomodulate PGE synthase activity of an isolated/purified polypeptide ofthe invention (including a full-length PGE synthase or an active portionthereof).

A method of screening for a substance which modulates activity of apolypeptide may include contacting one or more test substances with thepolypeptide in a suitable reaction medium, testing the activity of thetreated polypeptide and comparing that activity with the activity of thepolypeptide in comparable reaction medium untreated with the testsubstance or substances. A difference in activity between the treatedand untreated polypeptides is indicative of a modulating effect of therelevant test substance or substances.

In a further aspect of the invention there is provided an assay methodwhich includes:

(a) incubating an isolated polypeptide which has PGE synthase activityand a test compound in the presence of reduced glutathione and PGH₂under conditions in which PGE is normally produced; and

(b) determining production of PGE.

PGH₂ substrate for PGE synthase may be provided by incubation ofcyclooxygenase and arachidonic acid, so these may be provided in theassay medium in order to provide PGH₂.

Furthermore, PGE synthase catalyses sterospecific formation of 9-keto,11α hydroxy prostaglandin from the cyclic endoperoxide and so othersubstrates of PGE synthase may be used in determination of PGE synthaseactivity, and the effect on that activity of a test compound, bydetermination of production of the appropriate product.

Substrate Product PGH₂ PGE₂ PGH₁ PGE₁ PGH₃ PGE₃ PGG₂ 15 (S) hydroperoxyPGE₂ PGG₁ 15 (S) hydroperoxy PGE₁ PGG₃ 15 (S) hydroperoxy PGE₃

Thus, a more general aspect of the invention provides an assay methodwhich includes:

(a) incubating an isolated polypeptide which has PGE synthase activityand a test compound in the presence of a cyclic endoperoxide substrateof PGE synthase under conditions in which PGE synthase normallycatalyses conversion of the cyclic endoperoxide substrate into a productwhich is the 9-keto, 11α hydroxy form of the substrate; and

(b) determining production of said product.

As noted, the substrate may be any of those discussed above, or anyother suitable substrate at the disposal of the skilled person. It maybe PGH₂, with the product then being PGE.

An inhibitor of PGE synthase may be identified (or a candidate substancesuspected of being a PGE synthase inhibitor may be confirmed as such) bydetermination of reduced production of PGE or other product (dependingon the substrate used) compared with a control experiment in which thetest compound is not applied.

Product determination may employ HPLC, UV spectrometry, radioactivitydetection, or RIA (such as a commercially available RIA kit fordetection of PGE). Product formation may be analysed by gaschromatography (GC) or mass spec. (MS), or TLC with radioactivityscanning.

Combinatorial library technology (Schultz, JS (1996) Biotechnol. Prog.12:729-743) provides an efficient way of testing a potentially vastnumber of different substances for ability to modulate activity of apolypeptide.

The amount of test substance or compound which may be added to an assayof the invention will normally be determined by trial and errordepending upon the type of compound used. Typically, from about 0.1 nMto 10 μM concentrations of a test compound (e.g. putative inhibitor) maybe used. Greater concentrations may be used when a peptide is the testsubstance.

Compounds which may be used may be natural or synthetic chemicalcompounds used in drug screening programmes. Extracts of plants whichcontain several characterised or uncharacterised components may also beused.

Other candidate inhibitor compounds may be based on modelling the3-dimensional structure of a polypeptide or peptide fragment and usingrational drug design to provide potential inhibitor compounds withparticular molecular shape, size and charge characteristics.

Following identification of a substance which modulates or affectspolypeptide activity, the substance may be investigated further.Furthermore, it may be manufactured and/or used in preparation, i.e.manufacture or formulation, of a composition such as a medicament,pharmaceutical composition or drug. These may be administered toindividuals.

Thus, the present invention extends in various aspects not only to asubstance identified as a modulator of polypeptide activity, inaccordance with what is disclosed herein, and a substance obtained by amethod of the invention, but also a pharmaceutical composition,medicament, drug or other composition comprising such a substance, amethod comprising administration of such a composition to a patient,e.g. for treatment (which may include preventative treatment) ofinflammation or a cellular growth abnormality or other disease orcondition as discussed, use of such a substance in manufacture of acomposition for administration, e.g. for treatment of inflammation or acellular growth abnormality or other disease or condition as discussed,and a method of making a pharmaceutical composition comprising admixingsuch a substance with a pharmaceutically acceptable excipient, vehicleor carrier, and optionally other ingredients.

A substance identified using as a modulator of PGE synthase activity maybe peptide or non-peptide in nature. Non-peptide “small molecules” areoften preferred for many in vivo pharmaceutical uses. Accordingly, amimetic or mimic of the substance (particularly if a peptide) may bedesigned for pharmaceutical use. The designing of mimetics to a knownpharmaceutically active compound is a known approach to the developmentof pharmaceuticals based on a “lead” compound. This might be desirablewhere the active compound is difficult or expensive to synthesise orwhere it is unsuitable for a particular method of administration, e.g.peptides are not well suited as active agents for oral compositions asthey tend to be quickly degraded by proteases in the alimentary canal.Mimetic design, synthesis and testing may be used to avoid randomlyscreening large number of molecules for a target property.

There are several steps commonly taken in the design of a mimetic from acompound having a given target property. Firstly, the particular partsof the compound that are critical and/or important in determining thetarget property are determined. In the case of a peptide, this can bedone by systematically varying the amino acid residues in the peptide,e.g. by substituting each residue in turn. These parts or residuesconstituting the active region of the compound are known as its“pharmacophore”.

Once the pharmacophore has been found, its structure is modelled toaccording its physical properties, e.g. stereochemistry, bonding, sizeand/or charge, using data from a range of sources, e.g. spectroscopictechniques, X-ray diffraction data and NMR. Computational analysis,similarity mapping (which models the charge and/or volume of apharmacophore, rather than the bonding between atoms) and othertechniques can be used in this modelling process.

In a variant of this approach, the three-dimensional structure of theligand and its binding partner are modelled. This can be especiallyuseful where the ligand and/or binding partner change conformation onbinding, allowing the model to take account of this the design of themimetic.

A template molecule is then selected onto which chemical groups whichmimic the pharmacophore can be grafted. The template molecule and thechemical groups grafted on to it can conveniently be selected so thatthe mimetic is easy to synthesise, is likely to be pharmacologicallyacceptable, and does not degrade in vivo, while retaining the biologicalactivity of the lead compound. The mimetic or mimetics found by thisapproach can then be screened to see whether they have the targetproperty, or to what extent they exhibit it. Further optimisation ormodification can then be carried out to arrive at one or more finalmimetics for in vivo or clinical testing.

Mimetics of substances identified as having ability to modulatepolypeptide activity using a screening method as disclosed herein areincluded within the scope of the present invention. A polypeptide,peptide or substance able to modulate activity of a polypeptideaccording to the present invention may be provided in a kit, e.g. sealedin a suitable container which protects its contents from the externalenvironment. Such a kit may include instructions for use.

Further aspects and embodiments of the present invention will beapparent to those skilled in the art. The following experiments providesupport for and exemplification by way of illustration of aspects andembodiments of the invention.

All documents mentioned in this specification are incorporated byreference.

Experimental

As noted above, prior to the inventors' work disclosed herein there wasno suggestion that the cDNA sequence in the GenBank database labelled asMGST1-L1 (GenBank accession number AF027740) as well as a p53 inducedPIG12 (GenBank accession number AF010316 encodes human PGE synthase.Polyak et al. (supra.) noted merely that the PIG12 cDNA appeared toencode another, one might say yet another, microsomal glutathioneS-transferase.

The inventors expressed the protein identified by them as PGE synthase(SEQ ID NO: 2) in a bacterial expression system, employing the codingsequence of SEQ ID NO: 1. Following heterologous expression in E. coli,both cytosolic and membrane fractions were prepared. A rabbit antiserumwas raised against an internal peptide of PGE synthase and Western blotanalysis specifically detected a 15 kDa protein in the membrane fractionfrom bacteria expressing PGE synthase.

The bacterial membrane and cytosolic fractions were incubated with PGH₂in the presence or absence of reduced glutathione. The products(PGF_(2α), PGE₂, PGD₂ and 12-HHT) were analyzed by RP-HPLC using UVabsorption at 195 nm as well as on line radioactivity detection. Themembrane but not the cytosolic fraction was found to possess highglutathione dependent PGE synthase activity (0.25 μmol/min/mg).

A549 cells were used as a model to study cyclooxygenase-2 induction byinterleukin-1β. When A549 cells were grown in the presence ofinterleukin-1β (1 ng/ml) for 24 h a significant induction of the PGEsynthase protein was observed using Western blot analysis. Also, theantiserum specifically recognized a 16 kDa protein in commerciallyavailable partly purified PGE synthase activity isolated from sheepseminal vesicles.

Material and Methods

Materials

Rabbit anti-human PGE synthase antiserum was raised to the followingsynthetic peptide: CRSDPDVERSLRAHRN (SEQ ID NO: 3) conjugated withkeyhole-limpet hemocyanin (Innovagen, Lund, Sweden). This peptideantigen corresponds to amino acids 59-74 of PGE synthase (note: Cys 68was replaced with Ser, in case inclusion of Cys interfered with thepeptide synthesis).

Horseradish peroxidase-linked donkey anti-rabbit antibody was purchasedfrom Amersham Pharmacia Biotech, England. Film (hyperfilm ECL) was alsoobtained from the same source. Oligonucleotides were from Kebolaboratory, Sweden. Pfu DNA polymerase was from Stratagene, CA, USA.PGH₂ and 3H-PGH₂ was purchased from Cayman chemical, USA. PGF_(2α),PGE₂, PGD₂ and 12-HHT was obtained from Biomol, PA, USA. Glutathione andInterleukin-1β were from Sigma-Aldrich, Inc. HPLC solvents were fromRathburn Chemicals, Scotland. Partly purified PGE synthase, isolatedfrom ovine seminal vesicles, was obtained from Oxford BiomedicalResearch, Inc., MI, USA. The cell line A549 was from BoehringerIngelheim Biowhittaker, Belgium. Cell culture media and antibiotics werefrom Gibco BRL, Life Technology, Sweden. Protease inhibitor mixture,Complete™, was obtained from Boehringer Mannheim Scandinavia, Sweden.

Isolation and Cloning of PGE Synthase

The EST clone 143735 with GenBank accession number R76492, haspreviously been identified as “microsomal glutathione-S-transferase1-like 1” (MGST1-L1) encoded by GenBank accession number AF027740. Thesame gene product has also been characterized as “p53 induced PIG12”encoded by GenBank accession number AF010316.

The coding sequence of PGE synthase corresponding to the nucleotidesequence 19 to 477 of the EST clone 143735 (accession number AF027740)was amplified by PCR. Oligonucleotide primers were constructed toincorporate suitable restriction sites (Nde I-Hind III) into the 5′ and3′ ends of the product.

Primer 1 (sense): 5′-GAGAGA{umlaut over (C)}Ä{umlaut over (T)}Ä{umlautover (T)}{umlaut over (G)}CCTGCCCACAGCCTG-3′ (underlining is NdeI-site);

Primer 2 (antisense): 5′-GAGAGAÄÄ{umlaut over (G)}{umlaut over(C)}{umlaut over (T)}{umlaut over (T)}CACAGGTGGCGGGCCGC-3′ (underliningis Hind III-site).

In both primers GAGAGA are just additional flanking nucleotides.

PCR was performed with 0.2 mM dNTPs, 0.5 μM of the respective primer, 70ng template, 2.5 U of Pfu polymerase in 1×Pfu buffer (supplied by themanufacturer). The temperature cycles were 45 s at 94° C., 45 s at 60°C. and 45 s at 72° C., repeated 25 times. However, the first denaturingperiod was 4 min and the last extension period was 10 min. The PCRproduct was isolated by agarose gel electrophoresis, purified from thegel and cut with Nde I and Hind III.

The resulting product was gel-purified and ligated into the bacterialexpression vector pSP19T7LT (Weinander, R., et al.(1995) BiochemicalJournal 311, 861-6). Ligated plasmids were transformed into DH5aTMcompetent cells. Plasmids were isolated from a number of clones andcleaved with NdeI and Hind III followed by agarose gel electrophoresisto confirm the size of inserts. Selected inserts were sequenced on anApplied Biosystems 373A automated DNA sequencer using a dye terminatorcycle sequencing kit.

The expression construct containing the correct coding sequence for thePGE synthase was transformed into E. coli BL21 (DE3) (that harbored theplasmid pLys SL (Studier, F. W. (1991) Journal of Molecular Biology 219,37-44). Glycerol stocks were prepared and stored frozen at −70° C. forsubsequent use as starting material for the expression experiments.

Expression in E coli

Small aliquots (1-2 μl) of bacterial glycerol stock were grown in 2×YTovernight at 37° C. The cultures were diluted 1:100 into 2 l of TerrificBroth medium containing ampicillin (75 μg/ml) and chloramphenicol (10μg/ml) in a 5 l flask placed in a thermostated water bath. The culturewas oxygenated by air bubbling and grown until the OD₆₀₀ was 0.4-1.2. Atthis point expression was induced by the addition of 1 mM isopropylβ-D-thiogalactopyranoside, the temperature was switched to 30° C. andthe culture allowed to grow for another 4 h.

Thereafter, cells were pelleted and resuspended in 100 ml TSEG buffer(15 mM Tris-HCl, pH 8.0, 0.25 M sucrose, 0.1 mM EDTA, 1 mM glutathione).Lysozyme was added to a final concentration of 0.2 mg/ml, and themixture was gently stirred for 30 min at 4° C. Then the cells were lysedby six 30 s sonication pulses from a MSE Soniprep 150 sonifier at 40-60%of maximum power. Cell debris was removed by centrifugation at 5000×gfor 10 min. The supernatant was then centrifuged at 250,000×g for 1 hand the membrane pellets were finally resuspended in 10 mM potassiumphosphate, pH 7.0, 20% glycerol, 0.1 mM EDTA, 1 mM glutathione. Totalprotein concentration was determined by the Coomassie protein assayaccording to the manufacturer's instructions (Bio-Rad).

SDS PAGE and Western Blotting

Samples were diluted and boiled for two min in SDS-containing samplebuffer (Laemmli, U. K. (1970) Nature 227, 680-5). Proteins wereseparated through 14% polyacrylamide gels (Novex) and electroblotted(Towbin, H., et al.(1979) PNAS USA 76, 4350-4) onto PVDF (Pall)membranes. Transfer efficiency was visualized using pre-stainedstandards (Novex). Membranes were then soaked for 1 h at 25° C. inTris-buffered saline (100 mM Tris-HCl, pH=7.5, 150 mM NaCl) containing0.1% (v/v) Tween and 5% (w/v) non-fat dried milk. The membrane wassubsequently washed twice in 0.1% T-TBS followed by 1 h incubation at25° C. with the indicated antiserum (1:2000 dilution) in 0.05% (v/v)T-TBS and 2% (w/v) non-fat dried milk. Following several washing steps(2×1 min, 1×15 min and 3×5 min, the blot was incubated for 1 h at 25° C.with a horseradish peroxidase-linked donkey anti-rabbit antibody (1:2000dilution) in 0.05% (v/v) T-TBS and 2% (w/v) non-fat dried milk. Thewashing steps were repeated and subsequently enhanced chemiluminescencedetection was performed according to the manufacturer's instructions(ECL plus, Amersham Pharmacia Biotech, England).

Cell Culture

A549 cells were cultured in RPMI 1640 supplemented with heat-inactivatedfetal bovine serum (10%), fungizone (2.5 μg/ml), penicillin (100 U/ml),streptomycin (100 μg/ml), at 37° C. in an atmosphere of 5% CO₂. Cellswere seeded at a concentration of 0.15×10⁶/ml in 75 cm² flasks. Afterthree days, confluence was reached and cells were washed in PBS twice,then detached using 1.5 ml 1×Trypsin/EDTA solution (GibcoBRL) for 15 minat 37° C. in an atmosphere of 5% CO₂. Thereafter 3 ml medium was addedto quench the trypsin and cells were further diluted and reseeded atappropriate number/cm² as just described.

In order to investigate the effect of IL-1β on PGE synthase expressionin A549 cells, 1×10⁶ cells in 5 ml medium were plated in 25 cm² flasksand incubated for 24 h. Subsequently, the cells were washed in PBS threetimes followed by addition of 5 ml RPMI 1640 medium containing fetalbovine serum (2%), β(1 ng/ml) and incubated for another 24 h. Forharvest, cells were washed in PBS twice, trypsinated in 0.5 ml1×Trypsin/EDTA solution for 15 min at 37° C. 1 ml culture media wasadded and cells were centrifuged at 500×g for 10 min followed by twowashes in 1 ml PBS. Cells were resuspended in 50 μl homogenizationbuffer consisting of potassium phosphate buffer (0.1 M, pH=7.4) and1×Complete™ protease inhibitor cocktail. Samples were sonicated 2×10 s,then 50 μl boiling 2×Laemmli buffer was added and the sample was boiledfor additional 2 min.

PGE Synthase Enzyme Assay

The protein sample was diluted in potassium inorganic phosphate buffer(0.1 M, pH=7.4) containing 2.5 mM reduced glutathione. The reaction(total volume =100 μl) was started by the addition of 10 μM PGH₂ with orwithout 0.1 μCi 3H PGH₂ and terminated with 60 μl acetonitrile/HCl,lowering the pH to 3.2. In order to determine the formation of eitherPGF_(2α), PGE₂ or PGD₂, an aliquot (60 μl) was analyzed byreverse-phase-HPLC combined with UV detection (195 nm) and/orradioactivity detection using an online β-RAM detector (Inus System,Inc.). The reverse-phase HPLC column was Nova-Pak C18 (3.9×150 mm, 4 μmparticle size) obtained from Waters. The mobile phase was water,acetonitrile and trifluoroacetic acid (70:30:0.007, by vol) with a flowrate of 1 ml/min.

For analysis of 12-HHT, a mobile phase of methanol, water and aceticacid (70:30:0,01, by vol) was used and UV detection at 236 nm. Theamounts of produced PGE₂ and 12-HHT were quantified by integration ofthe areas under the eluted peaks at 195 and 236 nm, respectively.

Results

Identification of PGE Synthase

The amino acid sequence of human PGE synthase (SEQ ID NO: 2) has aminoacid sequence identity of 38% with MGST1. In addition, MGST1 and PGEsynthase display similar hydropathy profiles and high pI:s (>10).

Expression of PGE Synthase

PGE-synthase was expressed using a bacterial expression system. In orderto demonstrate protein expression, a peptide antiserum was raisedagainst PGE synthase (aa segment 59-74).

Both the membrane and cytosolic fractions from bacteria expressing PGEsynthase were analyzed by SDS-PAGE and Western blot. As control, themembrane fraction from bacteria expressing ratMGST1 was included. In alllanes, 5 μg of total proteins were analyzed. Results were obtained usingantipeptide antiserum against PGE synthase, corresponding preimmuneserum, and the antipeptide antiserum diluted in the presence of 10⁻⁶ Mpeptide antigen. The exposure time was 2 min.

The antiserum recognized a 15 kDa band in the membrane fraction frombacteria expressing PGE synthase. This band was not found in thecorresponding cytosolic fraction. Furthermore, the antiserum did notrecognize ratMGST1 expressed using the same expression system.

The detection of PGE synthase was specific since antiserum diluted inthe presence of 1×10⁻⁶ M antigen (peptide) lost the capability to detectPGE synthase.

Prostaglandin E Synthase Activity

The membrane fraction (0.02 mg total protein/ml) isolated from bacteriaexpressing PGE synthase was incubated for 2 min in the presence of PGH₂(10 μM including 1 μCi 3H PGH₂) and reduced glutathione (2.5 mM).

Reverse-phase HPLC chromatograms were plotted for the products formedafter incubations with PGH₂ (plotting counts per minute (CPM) againsttime in minutes).

FIG. 1A shows results obtained with PGE synthase membrane fraction mixedwith stop solution.

FIG. 1B shows results obtained with buffer.

FIG. 1C shows results obtained with PGE synthase membrane fraction. Thematerial in the experiments for which results are shown in FIG. 1B andFIG. 1C were incubated for 2 min prior to addition of stop solution.Products were detected using radioactivity detection. The first 20 minrepresents isocratic elution using water, acetonitrile andtrifluoroacetic acid (70:30:0.007, by vol) as mobile phase with a flowrate of 1 ml/min. Then a linear gradient was applied from 100% mobilephase to 100% methanol over a 10 min period, which was sustained for therest of the run.

FIG. 1C shows the RP-HPLC profile of radioactive labeled products formedunder these conditions. One major peak is produced eluting at 12.3 mincorresponding to the elution time of synthetic PGE₂. The material ofthis peak was collected, derivitized and analyzed by GC/MS confirmingits identity as PGE₂. A minor peak was also produced which eluted at31.5 min, corresponding to the retention time of 12-HHT.

The non-enzymatic formation of these products is shown in FIG. 1B. Inthis chromatogram the peak corresponding to PGE₂ accounts for less than25% of that formed in the presence of membranes from bacteria expressingPGE synthase. Instead, the major product formed corresponds to 12-HHTeluting at 31.5 min as well as another minor peak with a retention timeof 14.9 min, corresponding to the retention time of PGD₂. Thechromatogram in FIG. 1A shows the zero time incubation when substratewas added to buffer containing the membrane fraction pre-mixed with stopsolution. Little PGE₂ was detected and the major product peakcorresponded to the retention time of 12-HHT.

The PGE₂ formation (FIG. 1C) was abolished if the membrane was boiledfor 2 min prior to incubation, demonstrating the enzymatic nature ofcatalysis. Also, no PGE synthase activity was observed using themembrane fraction obtained instead from bacteria expressing ratMGST1. Ifthe membrane fraction was treated with N-ethylmaleimide (1 mM) for 5 minprior to incubation, the enzyme activity was abolished. No activity wasdetected in the cytosolic fraction from bacteria expressing PGEsynthase. Moreover, no PGE synthase activity was observed in microsomesobtained from Sf9 cells expressing MGST2.

FIG. 2 demonstrates the production of PGE₂ as a function of proteinconcentration. In this experiment the various dilutions of the membranefraction obtained from bacteria expressing PGE synthase were incubatedin the presence of PGH₂ (10 μM) and GSH (2.5 mM) for 2 min. The PGE₂formation was analyzed and quantified by RP-HPLC and UV detection at 195nm as described in Materials and Methods above.

A linear relationship was found using protein concentrations up to 0.015mg/ml. Thereafter the slope rapidly declines due to almost completeconversion of added PGH₂ into PGE₂.

FIG. 3 demonstrates the time function of the PGE₂ production afterincubation of the membrane fraction (0.02 mg/ml) with PGH₂ (10 μM) inthe presence or absence of GSH (2.5 mM) . The membrane fraction obtainedfrom bacteria expressing PGE synthase (0.02 mg/ml) was incubated withPGH₂ for the indicated times in the presence (filled circles) or absence(open circles) of glutathione. Filled triangles represent non-enzymatic(buffer only) PGE₂ formation after incubation with PGH₂. The productformation was analyzed by RP-HPLC and PGE₂ was detected and quantifiedusing UV absorbance at 195 nm.

A linear relationship is obtained during the first 60s of incubation.Thereafter the slope of the curve declines due to substrate depletion.

FIG. 3 also shows that the activity is dependent on the presence ofglutathione. The specific activity under linear conditions (substractingbackground formation) was 600 pmol/1.2min/0.002 mg membrane fraction(i.e. 250 nmol/min/mg).

Induction of PGE Synthase by IL-1β

A549 cells have been used to study cox-2 induction and have also beenreported to significantly increase their PGE₂ release followingtreatment with interleukin-1β. In order to investigate whether or notPGE synthase may also be regulated by this cytokine, A549 cells werecultured for 24 h in the presence of 1 ng/ml IL-1β. Normal cells as wellas cells treated with IL-1β were subsequently analyzed for PGE synthaseexpression by SDS-PAGE followed by Western blotting.

Total protein corresponding to 0.2×10⁶ cells grown for 24 h in thepresence (1 ng/ml) or absence of IL-1β was fractionated by SDS-PAGE andtransferred to PVDF membrane. The membranes were incubated using eitherPGE synthase antiserum or PGE synthase antiserum containing theantigenic peptide (10⁻⁶ M). Also analyzed was the commercial available,partly purified, PGE synthase from sheep seminal vesicles (6 μg) as wellas the membrane fraction from bacteria expressing human PGE synthase (5μg).

PGE synthase was induced by IL-1β. In the lane loaded with IL-1β treatedcells, a 15 kDa band appeared comigrating with the expressed PGEsynthase in bacteria. The recognition was lost if antiserum was mixedwith the antigenic peptide (1 μM), demonstrating specific detection ofPGE synthase in A549 cells treated with IL-1β. Significantly loweramounts of PGE synthase were detected in non-treated A549 cells.

Identification of an Immunoreactive 16 kDa Protein in Partly PurifiedPGE Synthase Derived From Ovine Seminal Vesicles

The commercially available impure PGE synthase preparation, partlypurified from ram seminal vesicles, was tested for cross reactivity tohuman PGE synthase antiserum.

From the results of the Western blotting, it was evident that a 16 kDaprotein band appeared in the lane loaded with 6 μg of this sample. Theband was lost using the peptide absorbed antiserum suggesting specificrecognition. The protein differs somewhat in size and appears morediffuse, which may suggest some kind of posttranscriptionalmodification.

PGE Synthase Activity Assay

Earlier studies have demonstrated that prostaglandins can be separatedby RP-HPLC and detected by UV spectrophotometry (Terragno et al., (1981)Prostaglandins 21(1), 101-12; Powell (1985) Anal. Biochem. 148(1),59-69). The molar extinction coefficient of PGE2 is 16,500 at 192.5 nm(Terragno et al., (1981) Prostaglandins 21(1), 101-12). The differencesbetween the absorbance at 192.5 nm and 195 nm was marginal (Terragno etal., (1981) Prostaglandins 21(1), 101-12). However, our results usingthe RP-HPLC conditions (described below) demonstrated a significantlymore stable baseline, with less noise at the higher wavelength. The mainproducts of PGH2 are PGF2α, PGE2 and PGD2. Using the described RP-HPLCconditions, the retention times were 19.0, 23.8 and 28.6 minutes forPGF2α, PGE2 and PGD2, respectively. In order to obtain an internalstandard we have tested 11β-PGE2 and 16,16-dimethyl PGE2. The lattercompound was too hydrophobic and could not be used in the describedisocratic system. In contrast, 11β-PGE2 eluted with a retention time of25.3 min with almost baseline separation from PGE2. In order toinvestigate the UV-absorbance relationship between 11-β PGE₂ and PGE₂,equal amounts (quantified by GC-MS) were analyzed by RP-HPLC andanalyzed by UV-absorbance at 195 nm. The two compounds showed identicalUV-absorbance properties. In order to test the recovery andreproducibility of solid phase extraction, known amounts of 11-β PGE₂and PGE₂ were diluted in sample buffer and acidified by adding the stopsolution (containing no iron chloride) followed by the addition ofacetonitrile (33% final cone) and subjected to analysis (10% (v/v) oftotal sample). Alternatively, after the addition of stop solution, thesample was extracted by solid phase extraction and the correspondingfraction (10% (v/v) of total sample) was then analysed. The amounts of11-β PGE₂ and PGE₂ before and after extraction were compared and therecovery was estimated to be 85-90%.

In order to quantify PGE₂, a standard curve of PGE₂ was made. The curvewas linear over the range from 0.9 pmol to 706 pmol (R²=0.9997,k=0.0012). For quantification we routinely use both the externalstandard as well as the internal standard technique, the latter methodaccounting also for losses during preparation.

Some difficulties may be encountered when assaying PGE synthase withPGH2. The substrate is very labile and decomposes non-enzymatically,with a half-life of about 5 min at 37° C., into a mixture of PGE2 andPGD2 with a E/D ratio of abut 3 (Hamberg et al., (1974) Proc. Natl.Acad. Sci. (USA) 71, 345-349; Nugteren and Christ-Hazelhof (1980) InAdv. in Prostaglandin and Thromboxane Res. 6, edited by B. Samuelsson,P. W. Ramwell, and R. Paoletti. Raven Press: NY, 129-137). Also, the PGEsynthase catalysis is very fast, which is why substrate depletion easilycan occur within seconds thus preventing a quantitative analysis. Afterthe reaction has been terminated, any remaining PGH2 must also rapidlybe separated from the products in order not to interfere with theresults. To cope with these properties of the substrate, the assay maybe performed as follows.

In order to minimize non-enzymatic production of PGE₂, the substrate(PGH₂) was always kept on CO₂-ice (−78° C.) until use and the enzymereaction was performed at 0° C. in the presence of PGH2 and reducedglutathione (GSH). A stop-solution was used, containing FeCl₂₁ whichconverted any remaining PGH₂ into HHT (Hamberg and Samuelsson (1974)Proc. Natl. Acad. Sci. (USA) 71(9), 3400-4). Also, the products are muchmore stable in organic solvents (Nugteren and Christ-Hazelhof (1980) InAdv. in Prostaglandin and Thromboxane Res. 6, edited by B. Samuelsson,P. W. Ramwell, and R. Paoletti. Raven Press: NY, 129-137), so weimmediately extracted the sample after termination by solid phaseextraction and kept the eluate in acetonitrile.

Assay Method

Protein samples were diluted in potassium inorganic phosphate buffer(0.1M, pH 7.4) containing 2.5 mM reduced glutathione (GSH). 4 μl PGH₂,dissolved in acetone (0,284 mM) was added to eppendorf tubes and kept onCO₂-ice (−78° C.). Prior to the incubation, both the substrate andsamples were transferred onto wet-ice (or 37° C.) for 2 min temperatureequilibration. the reaction was started by the addition of the 100 μlsample to the tubes containing PGH₂. The reaction was terminated by theaddition of 400μl stop solution (25 mM FeCl₂50 mM citric acid and 2.7 μM11-β PGE₂), lowering the pH to 3, giving a total concentration of 20 mMFeCl₂, 40 mM citric acid and 2.1 μM 11-β PGE₂. Solid phase extractionwas performed immediately using C18-chromabond columns. The samples wereeluted with 500 μul acetonitrile and thereafter 1 ml H₂O was added. Inorder to determine the formation of PGE₂ and 11-β PGE₂, an aliquot (150μl) was analyzed by RP-HPLC, combined with UV detection at 195 nm. Thereverse-phase HPLC column was Nova-Pak C18 (3.9×150 mm, 4 μm particlesize) obtained from Waters and the mobile phase was water, acetonitrileand trifluoroacetic acid (72:28:0.007, by vol). The flow rate was 0.7ml/min and the products were quantified by integration of the peakareas.

Discussion

MGST1-L1 was identified as a homologue to MGST1 exhibiting similaritiesboth on the sequence level (38% amino acid identity) as well asstructural properties (hydrophobicity profile).

MGST1-L1 was expressed using a bacterial expression system. When testedfor PGE synthase activity, membranes from bacteria expressing MGST1-L1exhibited a significant PGE synthase activity (0.25 μmol/min/mg)corresponding to the highest levels of normally occurring PGE synthaseactivity, i.e. in microsomes isolated from sheep seminal vesicles(Moonen, P., et al. (1982) Methods in Enzymology 86, 84-91) and ratductus deferens (Watanabe, K., et al.(1997) Biochemical & BiophysicalResearch Communications 235, 148-52). In fact, estimating that 1% of thebacterial membrane protein is MGST1-L1, the deduced specific activitybecomes 25 μmol/min/mg corresponding to a Kcat/Km in the 10⁶ M-1S-1range. Such high Kcat/Km values are hallmarks of extremely efficientenzymes (Fersht, A. (1985) Enzyme Structure and Mechanism (W. H. Freeman& Co., New York) Considering the short half-life of PGH₂ and theexistence of competing pathways it makes sense that a physiologicallyrelevant activity is highly efficient.

These results provide indication that MGST1-L1 is human PGE synthase,Western blot analysis confirmed successful expression of the protein andalso that PGE synthase protein expression was upregulated in A549 cells(a human lung adenocarcinoma cell line) following IL-1β treatment. A549cells have been used by many investigators to study the regulation ofcox-2 and related enzymes such as cytosolic phospholipase A2. Theresults agree with published data demonstrating the upregulation ofcox-2 and the several-fold increase of PGE₂ biosynthesis in response tointerleukin-1β treatment of A549 cells (Huang, M., et al.(1998) CancerRes. 58, 1208-1216; Mitchell, J., et al.(1994) British J. of Pharmacol.113, 1008-1014). Combined with these findings on cox-2, the data suggestthat PGE synthase and cox-2 are co-regulated and that PGE₂ biosynthesismay be dependent on the presence of both these enzymes. In accordance,an inducible PGE synthase activity has also been described inlipopolysaccharide-stimulated rat peritoneal macrophages, whichcoincides with cox-2 expression and changes the product formation infavour of PGE₂ (Naraba, H., et al.(1998) Journal of Immunology 160,2974-82; Matsumoto, H., et al.(1997) Biochemical & Biophysical ResearchCommunications 230, 110-4). The induction of PGE synthase (PIG12)following p53 expression in a colorectal cancer cell line (DLD-1)(Polyak, K., et al. (1997) Nature 389, 300-305) may also be ofimportance for understanding the role of cox and PGE synthase in cancerand apoptosis. Cyclooxygenase-2 has also been implicated in colon cancerthrough the beneficial effects observed by various NSAIDs on cancergrowth (Dubois, R., et al.(1998) Faseb J. 12, 1063-1073).

In summary, the first microsomal glutathione dependent PGE synthase hasbeen identified and characterised and demonstrated to be upregulated bythe proinflammatory interleukin-1β in a lung cancer cell line. Thiscytokine also upregulates cox-2 and cellular capacity to produce PGE₂.This provides PGE synthase as a novel target for drug development invarious areas, including inflammation, cancer and apoptosis, asdiscussed already above.

1. An isolated and pure polypeptide which is a PGE synthase and which comprises the amino acid sequence of SEQ ID NO.
 2. 2. An isolated and pure polypeptide which is a PGE synthase and which consists of an active portion of SEQ ID NO:2 wherein said active portion comprises amino acids 30-152 of SEQ ID NO.
 2. 3. An isolated and pure polypeptide which is a PGE synthase and which consists of an active portion of SEQ ID NO:2 wherein said active portion comprises amino acids 1-130 of SEQ ID NO.
 2. 4. An isolated and pure polypeptide which is a PGE synthase and which consists of an active portion of SEQ ID NO:2 wherein said active portion comprises amino acids 30-130 of SEQ ID NO.
 2. 5. The polypeptide according to claim 1 fused to a heterologous sequence of amino acids. 